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The interaction of human growth hormone with human lymphocytes from an established culture (IM-9) was studied using 125I-human growth hormone. The binding of 125I-human growth hormone was rapid; with human growth hormone at 0.1 nm a steady state was observed in 90 min at 30°. Bound labeled human growth hormone was dissociated rapidly by addition of excess unlabeled human growth hormone. Binding of 125I-human growth hormone to cultured lymphocytes was relatively insensitive to alterations in the pH and in the concentrations of Ca2+, Mg2+, or EDTA. At 30° there was very little degradation of labeled human growth hormone or of the specific receptor sites. Tryptic digestion destroyed the capacity of cells to bind human growth hormone. The IM-9 cells bound all human growth hormone preparations but not unrelated hormones or nonprimate growth hormones. The binding of 125I-human growth hormone was inhibited 10 to 14% with 1 to 2 ng per ml of unlabeled human growth hormone and 50% with 30 to 40 ng per ml, well within the range of hormone concentrations in vivo. Analysis of steady state data revealed a single order of binding sites with an affinity constant of 1.3 x 109 m-1 and about 4000 binding sites per cell. Numerous human growth hormone preparations were assayed by use of this receptor system as well as by immunoassay and by bioassay in vivo. The potencies of the human growth hormone preparations determined by the radioreceptor assay correlated more closely with the bioassay than with the radioimmunoassay.

SUMMARY Established human lymphoid cell lines bound calcitonin with high affinity. Calcitonin binding was detectable on both B cell and T cell lines derived from patients with a spectrum of lymphoproliferative disorders. These cell lines had marked differences in binding capacity per cell even when adjusted for differences in surface area. The binding was linearly related to cell density and showed a complex temperature dependency. Steady state kinetic analysis suggested a single class of high affinity receptors with an apparent association constant of 4 x lOlo MP. Competitive binding studies with calcitonin analogs revealed an order of Ki values similar to those previously obtained using plasma membranes from known calcitonin target tissues. Human calcitonin, as found previously with renal or skeletal membranes from the rat, competed only weakly for the lymphocyte receptors confirming that the human analog has evolved into a weak or unstable form, or both.

Fractions derived from whole bovine pituitary glands or bovine anterior lobes were found to inhibit the ovarian weight-gain activity of mixtures of ovine follicle stimulating hormone (FSH) and human chorionic gonadotropin (HCG), bovine FSH and HCG, or HCG alone. These fractions also inhibited the ovarian ascorbic acid depleting (OAAD) activity of HCG but not bovine luteinizing hormone (LH). Such effects were seen only when the gonadotropin inhibiting (GI) fraction was mixed with the gonadotropin prior to bioassay. Separate site injections of GI and gonadotropin abolished the inhibitory response. GI failed to inhibit endogenous secretions of gonadotropins in normal cycling female rats, as reflected by ovarian and uterine weights, ovarian histology and vaginal smear patterns. FSH and HCG, but not bovine LH, have been reported to require sialic acid for biologic activity. GI was shown to exhibit neuraminidase activity over a wide pH range. Because of this, it is suggested that a neuraminidase-like enzyme may...

Insulin interactions with human lymphocytes in established cultures and with isolated peripheral lymphocytes have been studied using 125I-insulin. Receptors in circulating lymphocytes are indistinguishable from those found in cultured cells, and the lymphocyte receptors show striking similarities to those structures described in fat cell and liver preparations. In lymphocytes, the ability of insulin or insulin analogues to inhibit binding of 125I-insulin is directly proportional to the ability of that preparation to inhibit binding of labeled hormone to purified liver membranes or to stimulate glucose oxidation in the fat cell. Binding of 125I-insulin to lymphocytes is a rapid and reversible process. Binding is maximal at 15°. Dissociation rate studies revealed a biphasic curve and the constants obtained were 8.3 x 10-5 s-1 and 8.3 x 10-4 s-1. The apparent affinity constants of the two orders of binding sites in the cultured lymphocytes were 1.2 x 1010 m-1 and 1.1 x 109 m-1, whereas in the normal circulating lymphocytes the values were 2.0 x 109 m-1 and 1.4 x 108 m-1. At 30° there is very little degradation of labeled insulin by lymphocytes, and at 15° there is no detectable degradation with cultured or circulating cells. Binding of 125I-insulin to cultured lymphocytes is unaffected by Ca++, Mg++, or EDTA. The optimum pH for binding occurs at about 7.8. Insulin binding to cells is unaffected by digestion with DNase, RNase, or neuraminidase. However, tryptic digestion destroys the capacity of the cells to bind insulin.

Specific insulin receptors from human lymphocytes in culture have been prepared using the nonionic detergent NP-40. Receptors were solubilized from intact cells and from crude plasma membrane fractions. Insulin receptors prepared in this manner retain many of the characteristics of the receptors studied in intact cells.

Summary A simple method is reported to detect cooperative interactions in the binding of polypeptide hormones to their membrane receptors. The dissociation of radioiodinated hormone from the receptor is studied under two conditions: first, by diluting the hormone-receptor complex sufficiently to prevent rebinding of the dissociated tracer; second, by dilution to the same extent in a medium containing an excess of unlabeled hormone. If the sites are independent, the dissociation rates must be the same in both cases. If the presence of unlabeled hormone increases the dissociation rate of the tracer, negatively cooperative interactions must occur. Insulin receptors on cultured lymphocytes and liver plasma membranes show negative cooperative interactions. Growth hormone receptor sites lack these interactions.

125I-insulin binds to circulating human lymphocytes and is displaced by unlabeled insulin. We have compared the binding and displacement curves of 125I-insulin from the circulating lymphocytes of normal subjects with those of insulin resistant obese and acromegalic patients and insulin sensitive hypopituitary subjects. The initial 125I-insulin binding (maximal percent 125I-insulin bound) for normal subjects had a mean value of 2.9 ng/ml (range 1.9–3.4) per 70 × 106 cells which is not statistically different from the other patient groups. For the normals, the [Insulin]-50% inhibition 125I-insulin bound (that concentration of insulin necessary to inhibit 50% of the maximal binding) had a mean value of 8.4 ng/ml (range 6.0–12.0 ng/ml). This value is significantly different from the obese patients but not from the acromegalic or the hypopituitary subjects. If the circulating lymphocyte mirrors the changes seen in the major metabolic sites of insulin action, these data suggest that an alteration in the insulin...

Chronic (5-16 hr) exposure of cultured human lymphocytes to 10 -8 M insulin at 37° in vitro produced a decrease in insulin receptor concentrations unaccounted for by simple occupancy of sites; acute exposure (0-2 hr) was without effect. These results reproduced observations in vivo where chronic hyperinsulinemia (e.g., 10 -8 M insulin in the circulation of obese insulinresistant hyperglycemic mice) is associated with a substantial reduction in the concentration of insulin receptors per cell, while acute hyperinsulinemia in vivo has no effect on receptor concentration. These data suggest a reciprocal relationship between insulin in the extracellular fluid and the concentration of insulin receptors per cell, which is mediated at the target cell itself by intracellular insulin-sensitive regulatory processes and directly affects target-cell sensitivity to hormone.

Specific insulin receptors from human lymphocytes in culture have been prepared in aqueous solution without use of detergents or related compounds. Receptors prepared in this fashion exhibit characteristics identical to those reported in intact cells.

Human lymphocytes obtained from fasted adult subjects and cultured human tumor lymphocytes were investigated for specific insulin receptors. By use of monoiodoinsulin, specific insulin binding sites were demonstrated in peripheral human lymphocytes, cultured human lymphocytes, and in other types of human circulating cells. Insulins and insulin derivatives that varied in their potency to stimulate glucose oxidation in the fat cell and to inhibit binding of [ 125 I]insulin to purified plasma membranes, varied in an analogous fashion in their ability to inhibit the binding of labeled insulin to human lymphocytes. Hormones that had no effect on the binding of insulin to fat cells or liver membranes also had no effect on the binding of insulin to lymphocytes. Binding was time and temperature dependent; dissociation of [ 125 I]insulin was rapid upon addition of 10 μM insulin. These findings afford a direct approach to the study of endocrine disorders in man.